SUMMARY OF WORK During the previous project period we have concentrated on the mechanisms involved in termination of local calcium release events. In collaboration with Eduardo Rios, we devised calcium spark simulations that were compared with high-resolution spark images obtained from mammalian skeletal muscle. This analysis showed that spark calcium sources are spatially extended and involve coordiated opening of large numbers of calcium release channels (ryanodine receptors, RyRs). We developed spark simulation algorithms in the FACSIMILE language which can be fitted to experimental records by non-linear least-squares methods. These simulations now take into account the possibility of local depletion of SR lumenal calcium, which has been suggested as a spark-termination mechanism. Our Monte Carlo simulations of local SR calcium release showed that the quantal decomposition of calcium sparks found by Dr. Cheng's group could be explained by very strong inactivation of of resting RyRs by local cytosolic calcium. However, to date, the existence of such a phenomenon is not confirmed by studies on isolated RyRs. We are therefore modifying the simulation algorithm to take into account dynamic local depletion of calcium in individual SR release terminals and regulation of RyR gating by lumenal Ca2+. This involves major alteration of the algorithm to make SR calcium a local dynamical variable. We will then determine whether the quantal statistics found experimentally can be accounted for by the lumenal calcium regulation mechanism. In other studies, we have modeled the effect of local spontaneous calcium release events on currents in pacemaker cells. The modeling shows that diastolic calcium release couples strongly via sodium-calcium exchange current so that SR calcium oscillations become a dominant mechanism in regulating heart rate. This is consistent with recent experimental results from our laboratory. We have also analyzed the first single-channel L-type calcium currents recored under physiological conditions and find that the mechanism of calcium-inactivation of the channel appears to be consistent with the mode-shift mechanism hypothesized in our stochastic simulations of excitation-contraction coupling.